The invention relates to an electrophoresis device and an electrophoresis method for the separation of macromolecules and the use of this device.
Electrophoresis devices are known for separating macromolecules from a substance, for example, separating polypeptides or nucleic acids, where the separation takes place according to the molecular weight or the structural characteristics.
Recently, methods have also become known which permit the separating of very large nucleic acid molecules or even intact chromosomal deoxyribonucleic acid (DNA), for example, of yeast cells (Saccharomyces cerevisiae). These methods include, for example, the "Pulsed Field Gel Electrophoresis" (PFGE) described, for example, in U.S. Pat. No. 4,473,452, and the "Orthogonal Field Alternation Gel Electrophoresis" (OFAGE) described by Carle & Olson in Nucl Acids Res. 12, 5647-5664, 1984).
It is also possible to use these methods for the analysis of chromosomal fragments, for example, of the human (Ragoussis et al, EBs Lett. 204, 1-4, 1986; Lawrance et al, Science 235, 1387-1390, 1987).
Both the PFGE and the OFAGE utilize the fact that large nucleic acid molecules apparently must first orient themselves in an electric field before they will be able to start travelling. The duration of this orientation phase is probably directly proportional to the length of the nucleic acid molecules (Smith et al, "Meth. Enzymol.", in the printing process, 1987). In the PFGE and the OFAGE, two non-homogeneous electric fields, alternating at an obtuse angle (via electronic circuits), act upon the macromolecules. As a result, in an alternating manner, the macromolecules must first reorient themselves in the one electric field and then in the second electric field before they can travel in the direction of the respective anode. However, a significant disadvantage of this method is the fact that the field intensities of the two non-homogeneous fields are identical only for the specimen located in the center. As a result, it is difficult to compare the mobilities of macromolecules in different specimens. In addition, according to these systems, the duration of a typical electrophoresis experiment is 40-72 hours (compare FIG. 1 in Lawrance et al, Science 235, 1387-1390, 1987).
Experiments addressing these difficulties are also known from literature. Chu et al (Science 234, 1582-1686, 1986) describe a modification ("Contour-Clamped Homogeneous Electric Fields") of the OFAGE which uses a hexagonal electrophoresis apparatus, the multiple electrodes of which are connected with one another via resistors. Although this electrophoresis method results in a good comparability of the mobility of macromolecules of different specimens, it requires extraordinarily large electrophoresis apparatuses when using the standard size (20.times.20 cm) agarose gels. These apparatuses are difficult to manufacture and also have considerable disadvantages with respect to cooling and the electric power supply.
Although in the case of a "Field Inversion Gel Electrophoresis" (Carle et al, Science 232, 65-68, 1986), the electrophoresis is carried out in conventional apparatuses with one cathode and one anode, the direction of the current is switched electronically from time to time. This results in the desired reorientation of the macromolecules. However, this method also has disadvantages because the dissolution process is not high, and the mobility of the nucleic acid molecules depends considerably on the concentration. In addition, the platinum electrodes corrode very rapidly.
A method is also known in which the gel, together with the macromolecules to be separated, rotate in a homogeneous electric field (Anand, Trends Genet. 2, 278-283, 1986). According to this method, it is difficult to steer the heavy gel table with the gel. Further, the required duration of these known electrophoresis methods is one weak and longer. Gels with very low agarose concentrations which allow the separation of DNA molecules with more than 1,000,000 base pairs, do not have the stability that is necessary for an efficient separation of the nucleic acid molecules in the course of this extreme duration.
An object of the present invention is to provide a device and a method which maintain the advantages of the methods discussed above, and which provide a particularly rapid and reproduceable separation of macromolecules from several specimens in a gel located in a buffer solution. Another object is to also provide an electrophoresis apparatus and method which provide an excellent comparability of the mobility without requiring expensive equipment for electronic switching processes or very costly electrophoresis apparatuses.
These objects and other objects are achieved by providing an electrophoretic separation apparatus suitable for the separation of macromolecules. The apparatus includes electrode elements which generate a electric field. The electrode elements can be rotated by rotating driving elements which rotate the electrode elements with respect to at least one specimen. A method corresponding to this apparatus is also provided.
This apparatus and method provide an excellent separation of macromolecules in an extremely short duration. Further, using the present invention, the electrophoresis device does not require an extremely large size as known apparatuses which have been used for the separation of macromolecular DNAs. This system is relatively inexpensive and is of simple construction and operation when compared to the known devices.
Other objects, advantages and novel features of the present invention will become apparent from the following detailed description of the invention when considered in conjunction with the accompanying drawings.